DNA polymerases synthesize DNA molecules in the 5' to 3' direction from deoxynucleoside triphosphates (nucleotides) using a complementary template DNA strand and a primer by successively adding nucleotides to the free 3'-hydroxyl group of the growing strand. The template strand determines the order of addition of nucleotides via Watson-Crick base pairing. In cells, DNA polymerases are involved in DNA repair synthesis and replication (Kornberg, 1974, In DNA Synthesis. W. H. Freeman, San Francisco).
Escherichia coli DNA polymerase I (DNApolI) and other homologous polymerases have three enzymatic functions: i) a 5' to 3' exonuclease activity, ii) a 3' to 5' exonuclease activity and iii) a DNA synthesis activity. The latter two functions are located towards the COOH-end of the protein, within a `Klenow` fragment (DNApolIK). Enzyme preparations of E. coli DNA pol I can be treated with subtilisin to yield a E. coli DNApolIK minus the 5' to 3' exonuclease activity (see Brown et al., 1982, J Biol. Chem. 257: 1965-1972; Joyce et al., 1982, J. Biol. Chem. 257: 1958-1964; Joyce et al., 1983, Proc Natl Acad Sci USA 80: 1830-1834; Klenow & Henningsen, 1970, Proc. Natl. Acad. Sci. USA 65: 168-175; Kornberg, 1974; Setlow, P. et al., 1972, J. Biol. Chem. 247: 224-231; Setlow and Kornberg, 1972, J. Biol. Chem. 247: 232-240; Steitz and Joyce, 1987, In Protein Engineering, Chap. 20, pp. 227-235. Oxender, D. A., and Fox, C. F. (eds). Alan R. Liss, New York). It has been shown that the DNApolI gene is unstable, although the DNApolIk gene has been cloned and is stable (Joyce et al., 1982, J. Biol. Chem. 257: 1958-64; Joyce et al., 1983, PNAS USA 80: 1830-34).
Research on E. coli DNApolIK has indicated that the sites of the 3' to 5' exonuclease and DNA synthesis activities may be separated as their corresponding substrates do not compete. The DNA synthesis active site binds to double-stranded DNA containing a single-stranded 5' extension and deoxynucleoside triphosphate (dNTP) whereas the 3' to 5' exonuclease active site binds to deoxynucleoside monophosphate (dNMP) (Ollis et al., 1985, Nature 313: 762-766). The existence of a conserved 3' to 5' exonuclease active site present in a number of DNA polymerases was predicted by Bernat et al., 1989, Cell 59: 219-228; Blanco et al., 1992, Gene 112: 139-144; Reha-Krantz, L. J., 1992, Gene 112: 133-137.
Crystal structure analysis of E. coli DNApolIK has shown that its peptide chain is folded into two distinct domains, with the smaller domain of 200 amino acid residues being the 3' to 5' exonuclease domain and the other domain of 400 amino acid residues being the DNA synthesis domain (Ollis, 1985).
Evidence using modified DNA substrates further supports the hypothesis that the 3' to 5' exonuclease domain is separate from the DNA synthesis domain (Cowart et al.,1988, Biochem. 20: 1973-1983). This view is also supported by base mutations and deletions in related T4 and T5 DNA polymerases (Frey et al., 1992, Proc. Natl. Acad. Sci. USA 90: 2579-2583; Leavitt et al., 1989, Proc. Natl. Acad. Sci USA 86: 4465-4469; Spacciapoli, P. and Nossal, N. G., 1994, J. Biol. Chem. 269: 438-446).
Both the 3' to 5' exonuclease and the DNA synthesis domains of E. coli DNA polymerase I have been cloned, expressed, and characterized independently. The 3' to 5' exonuclease domain consists of approximately 200 amino acids. The DNA synthesis domain contains approximately 400 amino acids. There is, however, 50-fold less activity in the DNA synthesis domain when compared to the E. coli DNApolIK (Morrison et al., 1991, Proc. Natl. Acad Sci. USA, 88: 9473-9477).
Bacillus stearothermophilus is a mesophilic bacterium. The DNApolI of Bacillus stearothermophilus displays optimum activity at 67.degree. C. Its amino acid sequence shows a high level of homology to that of E. coli polI. Other similarities include the presence of three active domains: 5' to 3' exonuclease, 3' to 5' exonuclease, and DNA synthesis (Kaboev et al., 1981, J. Bacteriol. 145: 21-26; Phang et al., 1995, Gene 163: 65-68; Stenesh and Roe, 1972, Biochim. Biophys. Acta 272: 156-166; Ye and Hong, 1987, Scientia Sinica 30: 503-506). Limited proteolysis with subtilisin also results in a small fragment with the 5' to 3' exonuclease activity and a large Klenow-like fragment (Lu et al., 1991, BioTechniques 11: 465-166; McClary et al., 1991, DNA Sequence 1: 173-180; Phange et al., 1995). The B. stearothermophilus DNApolI large Klenow-like fragement has been proposed to contain a domainc for the 3' to 5' exonuclease activity (Ye & Hong, 1987).
Many molecular cloning techniques and protocols involve the systhesis of DNA in in vitro reactions catalyzed by DNA polymerases. For example, DNA polymerases are used in DNA labelling and DNA sequencing reactions, using either 35S-, 32P- or 33P-labelled nucloetides. Most of these enzymes require a template and primer, and synthesize a product whose sequence is complementary to that of the template. The 5' to 3' exonuclease activity of E. coli DNA polymerases I is often troublesome in these reactions because it degrades the 5' terminus of primers that are bound to the DNA templates and removes 5' phosphates from the termini of DNA fragments that are to be used as substrates for ligation. The use of DNA polymerase for these labelling and sequencing reactions thus depends upon the removal of the 5' to 3' exonuclease activity.
The 5' to 3' exonuclease activity can be removed proteolytically from the holoenzyme without affecting either the polymerase activity of the 3' to 5' exonuclease activity. The klenow fragment of E. coli DNA polymerase I that is available. today from commercial sources consists of a single polypeptide chain produced by cleavage of intact DNA polymerase I with subtilisin or by cloning. This creates a DNApolIK containing only the 3' to 5' exonuclease and DNA synthesis activities (without the 5' to 3' exonuclease activity) and allows for net DNA processivity.
DNA processivity is performed by heat denaturation of a DNA template containing the target sequence, annealing of a primer to the DNA strand and extension of the annealed primer with a DNA polymerase. At low temperatures of about 37.degree. C., the DNA may be insufficiently denatured and secondary structures may impede DNA processivity. This results in stoppage of primer extension (insufficient labelling in DNA labelling) or errors (e.g., band compression or cross-banding artifacts in DNA sequencing). Band compression is artifacts seen on sequencing gels when there is a run of G-C rich regions. This may be overcome by adding deaza- or inosine-nucleotides to substitute for guanosine nucleotides or preferably by performing DNA processivity at elevated temperatures. Cross-banding artifacts are caused by premature termination, mismatches, or both.
The incorporation of a thermostable subtilisin-treated DNA polymerase, such as that from Bacillus stearothermophilus DNApolI, into DNA sequencing reactions allows for these secondary structure artifacts to be overcome. B. stearothermophilus DNApolI has a temperature optimum of 67.degree. C. and exhibits an ideal banding pattern with minimal or no crossbanding, due to the presence of a 3' to 5' exonuclease activity present in the enzyme. Crossbanding produces unreadable sequencing gels.
The subtilisin-digested B. stearothermophilus DNA polymerase I (DNApolI) is distinguished from other DNA polymerases by its high degree of DNA processivity and fidelity (Lu et al., 1991, BioTechniques 11: 465-466; McClary et al., 1991, DNA Sequence 1: 173-180; Mead et al., 1991, Biotechniques 11: 76-87; Ye & Hong, 1987, Sci. Sin. 30: 503-506). This is exemplified by readable DNA sequences on X-ray films of at least 350 bases with little or no cross-banding. In contrast, other DNA polymerases may have high degree of processivity but with poor fidelity, resulting in errors or cross-banding artifacts.
The high degree of polymerase activity is due to the DNA synthesis domain. The high fidelity conferred upon B. stearothermophilus DNApolI may be due to its proposed 3'-5' exonuclease activity (proofreading or editing function), which is proposed to reside independently of the DNA synthesis activity in a similar fashion to other Type I DNA polymerases as mentioned earlier; but Bst DNApolI may lack functional 3'-5' exonuclease activity (see Stellmann et al, infra).
Alternatively, high fidelity may not be due to the 3'-5' exonuclease activity, but rather, may be an inherent property of the DNA synthesis domain B. stearothermophilus; e.g., tighter binding of the enzyme to the DNA template may confer higher fidelity by preventing mismatches from occurring.
The concept of net DNA processivity, however, is the ratio of DNA synthesis activity versus 3'-5' exonuclease activity. DNA synthesis enzyme (synthetase) acts to polymerize nucleotides while 3'-5' exonuclease has an editing or proof-reading function. Thus high DNA synthesis is generally achieved at the expense of high fidelity and vice versa. The 3'-to-5' exonuclease activity of many DNA polymerases may, therefore, be disadvantageous in situations where one is trying to achieve net synthesis of DNA.
No document discussing DNA polymerase from B. stearothermophilus relates to a DNA polymerase enzme which exhibits reduced 3' to 5' exonuclease activity and which can give superior results in DNA amplification techinques and primer extention reactions. Further, prior documents do not appear to fully appreciate a genomic clone of Bacillus stearothermophilus, such as a gene or isolated nucleic acid molecule encoding a DNA polymerase from Bacillus stearothermophilus; Klenow-like fragments from expression thereof; or uses thereof, e.g., in manual or automated DNA sequencing and/or labeling.
There is thus a need for a B. stearothermophilus DNA polymerase I enzyme that has reduced 3' to 5' exonuclease activity or which is deficient in 3' to 5' exonuclease activity and can participate in various DNA amplifications schemes, including DNA sequencing and labelling, manual or automated. There is further a need for uses of the genomic clone of Bst, e.g., uses for nucleic acid molecules which encode a DNA polymerase from Bst, as well as for Klenow-like fragments therefrom and expression thereof; for example there is a need for producing DNA polymerases from Bst, and a need in manual or automated DNA sequencing and/or labeling for such polymerases.